4 General Cell Culture Methods
39
5. Centrifuge the cell suspension at 150 g for 5 min again.
6. Warm 45% PEG and the medium to 37°C.
7. Add 0.5 ml warmed PEG to the cell pellet over a period of 30 s, while
gently pipetting.
8. After 1 min, add 1 ml warmed serum-free Cp medium over a period
of 1 min, then make drop-wise additions of 8 ml of the same medium
over a period of 4 min.
1. Centrifuge the cell suspension at 150 g for 5 min.
2. Suspend the cells in growth medium, and seed them into the wells of
a hybridoma tissue culture tray.
3. Change half the medium on days 1 and 3 after cell fusion.
4. Continue culture in Cp medium at 27°C.
Results
According to Miltenburger et al. (1985), IZD-Cp 2202 cells showed 35%
viability at the time of cell fusion because of blocking of DNA and protein synthesis by inhibitors. Furthermore, less than 0.1 % of these cells
could recover without cell fusion. Approximately 2 weeks after cell fusion, cell colonies appeared in the hybridoma tray culture. From the
colony, a cell strain was obtained. This cell strain was thought of as a
hybrid of the IZD-Cp 2202 cell line and primary embryonic cells, because the authors could not get even one continuous cell line from the
sole primary culture of embryonic cells. Other characteristics, such as
DNA content, isozyme patterns, and virus susceptibility, supported the
hybrid nature of the cell strain obtained.
Comments
In general, it is not possible to produce a hypoxanthine-guaninephosphoribosyl transferase-deficient (HGPRT-) cell strain and thymidine kinase-deficient (TK-) cell strain in insect cell lines, and, therefore,
the HAT medium (hypoxanthine-aminopterine-thimidine-containing
medium) selection method to isolate hybrid cells cannot be used. Subsequently, Miltenburger et al. (1985) developed a rescue method for DNAand protein synthesis-blocked cells to isolate hybridized cells.
Subprotocol 6
Introduction of Oncogenes to Primary Cultured Cells to Obtain
Growing Cells
There are many primary cell cultures in which cells can survive for a
certain period, but not multiply. The administration of carcinogen to
such cells has been attempted to induce multiplication, without success.
Selection
of hybrid
cells
39
5. Centrifuge the cell suspension at 150 g for 5 min again.
6. Warm 45% PEG and the medium to 37°C.
7. Add 0.5 ml warmed PEG to the cell pellet over a period of 30 s, while
gently pipetting.
8. After 1 min, add 1 ml warmed serum-free Cp medium over a period
of 1 min, then make drop-wise additions of 8 ml of the same medium
over a period of 4 min.
1. Centrifuge the cell suspension at 150 g for 5 min.
2. Suspend the cells in growth medium, and seed them into the wells of
a hybridoma tissue culture tray.
3. Change half the medium on days 1 and 3 after cell fusion.
4. Continue culture in Cp medium at 27°C.
Results
According to Miltenburger et al. (1985), IZD-Cp 2202 cells showed 35%
viability at the time of cell fusion because of blocking of DNA and protein synthesis by inhibitors. Furthermore, less than 0.1 % of these cells
could recover without cell fusion. Approximately 2 weeks after cell fusion, cell colonies appeared in the hybridoma tray culture. From the
colony, a cell strain was obtained. This cell strain was thought of as a
hybrid of the IZD-Cp 2202 cell line and primary embryonic cells, because the authors could not get even one continuous cell line from the
sole primary culture of embryonic cells. Other characteristics, such as
DNA content, isozyme patterns, and virus susceptibility, supported the
hybrid nature of the cell strain obtained.
Comments
In general, it is not possible to produce a hypoxanthine-guaninephosphoribosyl transferase-deficient (HGPRT-) cell strain and thymidine kinase-deficient (TK-) cell strain in insect cell lines, and, therefore,
the HAT medium (hypoxanthine-aminopterine-thimidine-containing
medium) selection method to isolate hybrid cells cannot be used. Subsequently, Miltenburger et al. (1985) developed a rescue method for DNAand protein synthesis-blocked cells to isolate hybridized cells.
Subprotocol 6
Introduction of Oncogenes to Primary Cultured Cells to Obtain
Growing Cells
There are many primary cell cultures in which cells can survive for a
certain period, but not multiply. The administration of carcinogen to
such cells has been attempted to induce multiplication, without success.
Selection
of hybrid
cells